The correlation of gene and protein expression changes in biological systems has been hampered by the need for separate sample handling and analysis platforms for nucleic acids and proteins. In contrast to the simple, rapid, and flexible workflow of quantitative PCR (qPCR) methods, which enable characterization of several classes of nucleic acid biomarkers (e.g., DNA, mRNA, and microRNAs), protein analysis methods such as Western blotting are cumbersome, laborious, and much less quantitative. Proximity Ligation Assays (PLAs) have been shown to eliminate some of these problems. However, improvements to PLAs are desired by those of skill in the art.
Typical or conventional PLAs usually involve at least three or four steps. The first step is typically the binding of first and second probes (e.g., antibody probes) to a ligand (e.g., a protein of interest) such that the probes are in close proximity to another. Each of the probes typically contain an oligonucleotide. The oligonucleotides are brought into proximity to one another with the binding of the probes and, in the second step, are then ligated to one another (e.g., the ligation event). The ligated oligonucleotides may then be amplified and detected to determine the presence of the ligand with a test sample (e.g., a biological sample). This step is typically accomplished by adding ligation components, such as ligase, adenosine triphosphate (ATP) and buffer-salt mixture, to the binding reaction. In the third step, the ligase is typically then deactivated (e.g., by protease digestion) to prevent any further ligation of unbound oligonucleotides. In the fourth step, the reaction mixture is transferred to a real-time polymerase chain reaction (PCR) mixture and the quantity of the amplified product determined by quantitative PCR (qPCR). As described below, it has been surprisingly found that the third step (ligase digestion) may be eliminated, thereby allowing ligation and amplification to occur in the same reaction mixture without inactivation of the ligase. These and other features and advantages of the methods described herein will be apparent to the skilled artisan from this disclosure.